A brain-wide form of presynaptic active zone plasticity orchestrates resilience to brain aging in Drosophila

The brain as a central regulator of stress integration determines what is threatening, stores memories, and regulates physiological adaptations across the aging trajectory. While sleep homeostasis seems to be linked to brain resilience, how age-associated changes intersect to adapt brain resilience to life history remains enigmatic. We here provide evidence that a brain-wide form of presynaptic active zone plasticity (“PreScale”), characterized by increases of active zone scaffold proteins and synaptic vesicle release factors, integrates resilience by coupling sleep, longevity, and memory during early aging of Drosophila. PreScale increased over the brain until mid-age, to then decreased again, and promoted the age-typical adaption of sleep patterns as well as extended longevity, while at the same time it reduced the ability of forming new memories. Genetic induction of PreScale also mimicked early aging-associated adaption of sleep patterns and the neuronal activity/excitability of sleep control neurons. Spermidine supplementation, previously shown to suppress early aging-associated PreScale, also attenuated the age-typical sleep pattern changes. Pharmacological induction of sleep for 2 days in mid-age flies also reset PreScale, restored memory formation, and rejuvenated sleep patterns. Our data suggest that early along the aging trajectory, PreScale acts as an acute, brain-wide form of presynaptic plasticity to steer trade-offs between longevity, sleep, and memory formation in a still plastic phase of early brain aging.

10 progress in this work compared to previously published work from this group is unclear.

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First of all, we would like to express our deep gratitude to the reviewer for having carefully read and 12 commented on our manuscript in an insightful manner. We believe that this allowed for substantially improving 13 our revised manuscript. In response to the following comments, we performed a series of new experiments 14 and elaborated our interpretations and model.

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The hypothesis of behavioral adaptations executed by PreScale during early aging we propose here is based 16 on both biochemical and behavioral experiments. While studies mostly focus on advanced age, how the early 17 phase of aging affects behavior is often neglected, which is taken into consideration in this study. We propose 18 that early aging-associated behavioral alterations might well be adaptive and survival-protective. Indeed, 19 while our manuscript was under revision at PLOS Biology, a study published at Nature Aging show that 20 rapamycin treatment in early adulthood suffices to extend lifespan, but not during advanced aging [1].

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We previously proposed the causal role of PreScale in memory decline during early aging [2], and the causal

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We propose that PreScale is formed "on demand" in response to a neuronal physiological state switch which 28 builds in response to aging as well as to sleep deprivation. PreScale then coordinates to favor longevity over 29 new memory formation. Our data suggest that THIP treatment and spermidine supplementation are able to

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The solid curves represent normal aging, the right-shifted dashed curves are the aging process under spermidine (Spd) 36 supplementation or Gaboxadol (THIP) treatment.

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We provide the following new data in our revised manuscript: 2 3) We performed memory experiments showing that flies with advanced age (50-day-old) were no longer 3 improved by THIP treatment, compared to flies during early aging (30-day-old) (manuscript Figures 6Q-6R).

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These data support the model that early aging phase is likely adaptive, plastic and reversible, while advanced 5 aging phase might be non-plastic and non-reversible. 6 4) We examined the effects of 1xBRP on dFB and R5 utilizing CaLexA system (manuscript Figure S2).

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Please allow us to highlight the major advances of our current work (also including the data incorporated 9 during the revision) over our previous findings:  14 iii) We show that spermidine supplementation, previously shown to extend lifespan, promote memory 15 formation and suppress PreScale in mid-aged animals, does also attenuate the early-age-associated sleep 16 pattern changes (manuscript Figures 5 and S5) which we report in the current study. 17 iv) To decipher a role of age-associated sleep pattern changes alongside early aging, we used an acute 18 treatment with sleep-promoting Gaboxadol (THIP). We show that THIP treatment has similar consequences 19 as we have observed for spermidine supplementation: extended lifespan, improved memory formation and 20 suppressed PreScale (manuscript Figures 6 and 7). We further show that the suppressed PreScale is causal 21 for memory improvement after THIP treatment in mid-aged flies (manuscript Figure 7). Thus, both paradigms 22 seemingly converge into a similar scenario allowing for a reset along the early aging trajectory.

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Concerning the organization of our manuscript, we started by explicitly characterizing synaptic plasticity across 27 the fly lifespan (manuscript Figure 1), followed by cellular and mechanistic examination at the dFB neurons 28 for the comparison between genetic PreScale mimicry (4xBRP) and early aging-associated PreScale (in 20-29 day-old animals, manuscript Figure 2). Afterwards, we provided detailed analysis of the survival and 30 behavioral-relevance of PreScale during early aging (manuscript Figures 3 and 4). We then analyzed two 31 rejuvenation paradigms (spermidine/Spd supplementation and Gaboxadol/THIP treatment) in coupling with 32 PreScale-type plasticity and early aging-associated memory decline (manuscript Figures 5-7). We hope that 33 our manuscript is now more comprehensive and the idea that PreScale plays a central role in coupling sleep 34 homeostasis, memory and longevity is delivered more clearly.

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1) The Introduction is short and vague. The first two paragraphs, which make up the bulk of the

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We deeply appreciate this comment. In the revised manuscript, we thoroughly introduced our previous work  suggest that if PreScale is inhibited, sleep is reduced. On the other hand, if sleep is increased, PreScale is 3 inhibited. These seem to be opposing effects that the authors could discuss more clearly.

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We apologize for having being unclear and somewhat confusing at this point.

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For spermidine, we examined the consequences of its treatment in early aging-associated sleep pattern 6 changes, meanwhile memory is retained and PreScale is inhibited.

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For Gaboxadol/THIP, it is strongly sleep-promoting during acute treatment (manuscript Figure S6). Effects on 8 the sleep behavior of Drosophila after having applied THIP were not reported so far. We also examined the 9 consequences of THIP treatment (i.e. after THIP treatment). We now in the revised version show that indeed 10 30d wt flies exhibited less sleep when tested after THIP treatment, similar to the effects of spermidine 11 supplementation (Reviewer figure 2, now also incorporated into manuscript Figure 7). This effect is specific to 12 30d aged flies, as 5d flies did not show difference in sleep after THIP treatment (manuscript Figure S7).

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We now tried to graphically bundle our findings and interpretations in the proposed model (GRAPHIC

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We now arrive at a very similar relation concerning the effects of acute Gaboxadol/THIP and spermidine/Spd 16 supplementation in mid-aged 30d animals: suppressed PreScale, protected ability to form new memories, as 17 well as increased lifespan. We speculate that THIP treatment and spermidine supplementation might be able 18 to reset the neuronal physiological state during early aging and thus make PreScale dispensable (Reviewer

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The reviewer is definitely right that establishing causal relations in the aging context is challenging, also given 14 the interconnected nature of the phenotypic readouts. In this manuscript, we exploited and further established 15 a genetic mimicry of early aging-associated PreScale (by solely tuning brp gene copy number), which we found 16 to allow for phenocopying several aspects of sleep deprivation-and early aging-provoked changes in young 17 flies. Still, as correctly expressed by the reviewer, a challenge but also chance here is to establish epistatic 18 relations between this form of plasticity and the relevant behavioral components.

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To explicitly test an epistatic relation in the revision of our manuscript, we examined the effects of genetically 20 triggering PreScale (by increasing BRP copies from 2xBRP to 3xBRP) when interfering with the effects of THIP 21 treatment on memory at 30d. We reasoned that, if the suppression of PreScale (measured via brain BRP levels)

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was indeed causally connected with the post-THIP-observed memory improvement in 30d flies, increasing 23 BRP copies from 2xBRP to 3xBRP might attenuate the post-THIP memory effects. This is indeed what we found:

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THIP did improve memory in 2xBRP but not 3xBRP flies at the age of 30d (Reviewer figure 4). Notably, THIP 25 did not increase memory at 5d in neither 2xBRP nor 3xBRP (Reviewer figure 4). These data are now also   This is surely a valuable suggestion. Obviously, age-specific brp RNAi induction experiments need a post-10 developmental, temporally well-defined knockdown scenario without interfering a normal aging process. Thus,

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we decided to not use the Gal80 ts system, which depends on temperature shifts from 18°C to 29°C, a procedure 12 which change the aging process, sleep behavior and many other aspects. Instead, we in the context of this 13 revision performed a first experiment using the GeneSwitch system [4]. In our pilot experiments towards this 14 question, we used elav-GeneSwitch(GS)-Gal4 to induce brp RNAi at the age of 30d by feeding the flies with 15 the inducer RU486. As shown below, feeding 30d flies with 1mM RU486 for 5 days did provoke a slight trend

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This is a very interesting question. To answer this, we examined CaLexA signals in dFB and R5 of 1xBRP flies 26 at the age of 5d. While increasing BRP copies from 2x to 3x and 4xBRP upregulates R5 neuronal activity, 27 decreasing BRP copies from 2xBRP to 1xBRP did not change the activity of these neurons (Reviewer figure 6, 28 now also incorporated into manuscript Figure S2).

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Interestingly, we found that both increasing (3xBRP and 4xBRP) and decreasing (1xBRP) BRP copies reduced 30 the neuronal activity of the dFB (Reviewer figure 6, now also incorporated into manuscript Figure S2). We

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We thank the reviewer for suggesting these experiments. Unfortunately, patch clamp in vivo 18 electrophysiological recordings in the adult Drosophila brain are really challenging, especially for dFB neurons 19 whose cell bodies are particularly small. As also found in rodents, increasing age makes patch clamp recordings 20 even more challenging. As we described in the first submission, it was almost impossible to record 30-day-old 21 dFB neurons and, despite efforts, we only could record from a few 20-day-old animals in the original 22 submission (Please refer to the lines 168-171). It is unfortunately impossible to finish these experiments in a 23 time frame reasonable for the revision of our manuscript.

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As Reviewer #2 suggested to analyze the dFB electrophysiology of young 5d 3xBRP flies, we decided to start    regarding the conceptual framework, data interpretation, and experimental design. A primary concern is that 20 they do not explore sex differences in their data and do not examine reproductive fitness as a critical factor 21 in a fly's life. Moreover, the authors conclude that "a brain-wide form of presynaptic active zone plasticity 22 ("PreScale") promotes resilience by coupling sleep, longevity and memory during aging (abstract)." 23 However, while the manuscript provides correlative data, it is unclear whether they point to a causal role of 24 PreScale in the coordinated age-associated changes. Specific concerns are listed below.

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We thank this reviewer for in depth reviewing our work and finding it per se interesting. We deeply appreciate 26 the great suggestions concerning sex differences and reproductive fitness, as we also wanted to thoroughly 27 examine the roles of PreScale in various aspects of organismal aging.

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We admit that establishing causal relations in the aging context is challenging, also given the interconnected 29 nature of the phenotypic readouts. As mentioned above and in the revised manuscript, the genetic mimicry    1. In what sense is the tradeoff between longevity and memory during early aging optimal? What is 3 optimized? It seems that simply living longer wouldn't be the core purpose of a fly. One could argue that 4 producing lots of healthy progeny would be the fly's core mission. The authors should examine reproductive 5 fitness (e.g., # of offspring) as a critical aspect of the necessary tradeoffs as flies age.

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We sincerely thank the reviewer for this great suggestion. We now tested the fecundity of 1-4xBRP female 7 flies along aging. We found that 1xBRP, 2xBRP and 3xBRP flies were not different at most ages, except 50d 8 at which 1xBRP flies were obviously lower in the eggs produced per female (Reviewer figure 8). 4xBRP flies, 9 however, were always lower in the amount of eggs produced per female at each age tested (As shown in 10 manuscript Figure 3A, BRP level in 5d 4xBRP was already higher than reached in the context of normal aging).

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However, we consider the 3xBRP scenario as a physiological mimicry of the extent of normal early aging-12 associated PreScale (manuscript Figures 1 and 3A). As the fecundity of 3xBRP was not reduced versus 2xBRP 13 (but at the same time 3xBRP lives longer), 3xBRP animals might indeed be able to produce more offspring in 14 a lifetime (however, under challenging, variable conditions, 2xBRP with its more effective memory might be 15 advantageous). Thus, a moderate extent of PreScale during early aging (mimicked by 3xBRP) seems obviously 16 to not undermine fecundity (Reviewer figure 8). For the moment being, and given the somewhat limited extent 17 of our fecundity analysis, we would prefer to not include the data in the revised manuscript. However, if the 18 reviewer has different opinion on this decision, we would be prepared to include these data.

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Female flies were sorted into small vials with normal food 2 days in advance of their age indicated in Reviewer figure 8.

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Depending on the ages, the number of female flies within each vial was different. 3 female for age 3d, ~4 for 10d and 20d, ~6 for 23 30/40/50d. In each vial of the whole experiment, two 5d wt male flies were included. Sorted flies were transferred into fresh 24 normal food at zeitgeber time (ZT) 3, and were allowed to lay eggs for 24 hours. Afterwards, the amount of eggs in each vial was 25 manually counted. n = ~13 per group. One-way ANOVA with Bonferroni's multiple comparisons test is shown. ***p < 0.001.

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Here, under our normal stable laboratory rearing condition of aging process within which the lifespan is often 29 maximized, we propose that a balance between longevity and memory formation is optimized by PreScale, 30 likely through the regulation of sleep pattern changes during early aging. Living longer in stable laboratory 31 rearing conditions, while accompanying with compromised costly behaviors (e.g. memory formation) without 32 undermining reproductive success, could certainly allow for more egg production and finally more progenies 33 in a fly's life. In this sense, the trade-offs between memory and longevity executed by PreScale during early 34 aging without reducing reproductive fitness might actually be optimal. 35 2. The data show substantial sex differences (e.g., Fig. 3), but there is no serious discussion of the subject.

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As mentioned in Point #1, the authors should examine how reproductive success is impacted by various 37 genetic and drug manipulations.

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We appreciate this comment. We discussed the sex difference in the revised manuscript. Please refer to the  has adaptive advantages, why do spermidine and THIP, which suppress PreScale, improve both memory and 9 longevity? The authors suggest that Spd and THIP treatment promote efficient mitochondrial electron 1 transport and autophagy, making PreScale unnecessary. How can we tell a priori whether a particular PreScale 2 manipulation will enhance both memory and longevity or favor one over the other? What is meant by "a 3 specific, context-dependent role of PreScale in regulating lifespan (Line 182)." Please elaborate on the specific 4 contexts. If wake and inc mutants already have increased BRP, why does it help to have even more?

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We appreciate discussing these considerations and clarifying our perspective. We propose that Spd and THIP 6 treatment eliminate the need for the occurrence of PreScale during early aging, leading to the absence of 7 trade-offs between memory and longevity, e.g. both memory and longevity are promoted. From the extended 8 longevity and suppressed memory of 3xBRP animals (and the stable, unchanged fecundity of 3xBRP versus 9 2xBRP), it seems that PreScale primarily favors survival (and subsequently a likely somewhat higher 10 reproductive success) over "costly" memory [7,8]. Manipulations reducing PreScale (Spd supplementation 11 and THIP treatment) thus allowed to "reopen the window" for the formation of new memories (Reviewer figure   12 1 and manuscript Figure 8). It is certainly very interesting to understand how exactly Spd and THIP extend 13 lifespan without PreScale. We speculate that Spd and THIP might steer metabolic reprograming to regulate 14 lifespan.

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In Huang et al., 2020, we proposed that increasing BRP upon sleep loss in wake and inc mutants is a protective 16 action to counteract sleep loss (for example by promoting sleep). Introducing an additional brp copy thus help 17 the mutants in this direction.

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The statement "a specific, context-dependent role of PreScale in regulating lifespan" was based on the results that 3xBRP  4. The increased longevity due to PreScale and THIP treatment could be because they sleep more and therefore 23 spend less energy. The disadvantage may be that they do not have the opportunity to produce many offspring.

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Again, if the authors consider reproductive fitness, the increased longevity accompanied by increased sleep 25 may not be a good tradeoff.

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We thank the reviewer for triggering this discussion. As we also discussed in the original submission (lines  5. Fig. 2 shows striking physiological effects of increasing BRP levels to 4x. However, sleep and longevity data 33 suggest that whereas 3xBRP provides some beneficial effects similar to PreScale, 4xBRP is detrimental. Given 34 this finding, they should examine the electrophysiological properties for 3xBRP, not 4xBRP.

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We thank the reviewer for suggesting these experiments. Even though patch clamp in vivo electrophysiological 36 recordings in the adult Drosophila brain are really challenging, especially for dFB neurons whose cell bodies 37 are particularly small, we managed to patch a few 5d 3xBRP dFB neurons. These neurons were all in their OFF  Figures 2C and 2D).

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Taken together, as both 3xBRP and 4xBRP flies show increased sleep at age 5d [3] and they all show 47 significantly reduced CaLexA signal in dFB neurons (manuscript Figures 2C and 2D), we believe that 3xBRP 48 flies are likely similar to 4xBRP in dFB electrophysiology at young age 5d.

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Furthermore, we tend to believe that, at young age 5d when the mortality rate is as low as 2xBRP wt, 4xBRP

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OFF state dFB neurons lack of spontaneous spikes (A), and current injections did not elicit action potentials (B). Though we were 5 not able to analyze most of the electrophysiological parameters in OFF state dFB neurons, we observed a clear decrease in the 6 input resistance of 5d 3xBRP dFB neurons (C), similar to 4xBRP animals (manuscript Figure 2J). n = 3 per group. Student's t 7 test is shown. Error bars: mean ± SEM.

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We would like to emphasize that, the circuit formed by dFB-Helicon-R5 neurons is not isolated in the brain

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We also discussed these results in the revised manuscript (Please refer to the lines 478-490).

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7. In the model (graphical abstract and Fig. S7), they show "longevity" as something affected by aging. Since 22 longevity stays constant across the lifespan, mortality, which increases with aging, would be more helpful.

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Very good suggestions! We followed these thoughts in our revised manuscript. As for the model, we present 24 it in a more comprehensive and simplified manner in the revised manuscript (Graphical abstract and 25 manuscript Figure 8).

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8. The effects of THIP on memory seem variable across experiments. For example, 0.1THIP had no effect on 27 STM in one experiment (H) but a significant effect in another (N). The effects of 3xBRP on longevity are also 28 variable across experiments (Fig. 3C vs. E). What accounts for the differences?

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We are happy to clarify this point. STM in manuscript Figure 6H was tested immediately after 0.1THIP 30 treatment, while STM in manuscript Figure 6N was tested one day after 0.1THIP treatment. This is indeed an 31 observation we emphasized already in the original version (lines 319-323 and 326-327). Please also refer to 32 the difference in protocol in manuscript Figure 6E and Figure 6M.   We propose in the manuscript that early aging until middle age represents a still plastic phase and PreScale 7 adds on adaptivity here allowing to favor better survival over memory formation (Reviewer figure 1 and 8 manuscript Figure 8). However, after middle age with increased mortality rates, flies might enter into a non-9 plastic phase (Reviewer figure 1 and manuscript Figure 8). We suggest that in advanced age, additional factors 10 might become rate-limiting, e.g. metabolic reprogramming, mitochondrial function, and autophagy status.

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Indeed, to potentially answer this questions, we performed memory experiments on 50d wt flies after 2-day 12 THIP treatment, compared to 30d animals. We show that such a treatment can no longer reverse memory 13 defects in 50d flies (Reviewer figure 10). It will be very interesting to identify these additional factors and 14 processes limiting the reversibility of memory formation in advanced age in future studies.

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We appreciate this comment. We used both female and male flies for longevity and memory experiments.